In this project we are investigating the fundamental mechanisms of photodynamic therapy (PDT) action on tumor microstructure. Specifically, the subcellular localization and subcellular phototoxicity of PDT drugs are being investigated. Initially, CPAE endothelial and PTK2 epithelial cells were studied by visualizing the subcellular organelle fluorescence when the cells were exposed to 5-aminolaevulinic acid (ALA). Both cell types showed a large amount of fluorescence in the perinuclear cytoplasm, where the photosensitizer selectively localized in the mitochondria. To investigate the subcellular phototoxicity of ALA, the subcellular regions of the cells were irradiated with a 630 nm laser microbeam. The results demonstrated that the nucleus followed by the perinuclear cytoplasm are the most sensitive areas of the cell. Particularly intriguing was the finding that the nucleus was the most sensitive region since 1) no fluorescence was observed in the nucleus and 2) th e generally accepted mechanism for PDT damage involve singlet oxygen targeting of cytoplasmic elements. Based upon these findings we will further investigate the subcellular localization and phototoxicity of ALA and other PDT drugs using 2-photon excitation. Activation of PDT drugs through multiphoton absorption provides greater selectivity than single photon absorption. in addition, the 2-photon fluorescence will allow improved visualization of drug distribution within the cell, especially along the optical axis.